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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-07-12 to 1994-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
416-600-4
EC Name:
-
Cas Number:
77703-56-1
Molecular formula:
C23H32N4O2
IUPAC Name:
3-butyl-1-[4-({4-[(butylcarbamoyl)amino]phenyl}methyl)phenyl]urea
Specific details on test material used for the study:
Analytical purity: >= 99.65 %
Lot/batch No.: 93.166

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98, TA 1538) mutations.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 8 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 8 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulphoxyde (DMSO)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98, TA1537 without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-A)
Remarks:
TA98, TA100, TA1535, TA1537, TA1538 with S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
TA1535 without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without S9-mix
Details on test system and experimental conditions:
Preliminary study:
A preliminary study is performed on one bacterial strain TA100 (or TA98), without metabolic activation, in order to evaluate the toxicity of the test article to bacteria.
Increasing concentrations of the test article are used; examples for which are as follows: 8 - 40 - 200 - 1000 - 5000 mg/plate. The highest dose is 5000 µg/plate if the formulation type allows this dose level to be reached. A minimum of 5 dose levels are used.
Each concentration is tested in duplicate. At the end of the incubation period, the plates are observed for signs of toxicity. The colonies which appears in the presence of the test article are counted and the intensity of the bacterial lawn examined. These observations are compared with those performed in the presence of the vehicle. The signs of toxicity (reduction in bacterial lawn or reduction of the number of colonies) are noted.
Main study:
Two studies are conducted, the second study being performed to confirm or to complement the results of the first one for example by using a closer range near to the top limit dose.
For each study, each concentration of the test article is tested in triplicate with and without metabolic activation on each bacterial strain.
Concentration of the test substance resulting in precipitation: 1000 µg/plate
Evaluation criteria:
The test article will be considered to be clearly mutagenic if:
- The assay is valid
- The number of revertants in presence of the test article is greater than or equal to twice the number of revertants in the negative control at one dose level (the upper dose before toxicity) or more with a dose correlation
- The positive trends/effects are reproducible.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
NA

Any other information on results incl. tables

The test article PATE HAT was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA100 without metabolic activation.

Five concentrations (5000, 1000, 200, 40 and 8 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 2500, 1250, 625 and 313 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

Conclusion: Under the experimental conditions employed, it can be concluded that the test article PATE HAT did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed

Applicant's summary and conclusion

Conclusions:
HAT ISO was tested for mutagenic activity in the Ames-test with the bacterial strains S. typhimurium TA98, TA100,TA1535, TA1537, TA1538 with and without metabolic activation in two independent studies. No increase of the number of revertants per plate was observed. No classification and labelling for genetic toxicity is required according to Regulation 1272/2008/EC (CLP).
Executive summary:

PATE HAT was tested on 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) with and without metabolic activation.

Five concentrations (5000, 1000, 200, 40 and 8 µg/plate) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near the top of limit dose (5000, 2500, 1250, 625 and 313 µg/plate). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

PATE HAT did not cause an increase in the number of revertants per plate of any of the tester strains used, either in presence or absence of a metabolic activation system in both of the two independent studies performed